The present invention generally relates to methods and apparatus for characterization of polymer samples in liquid chromatography systems, and specifically, for characterization of polymer samples in multi-dimensional liquid chromatography systems. The invention particularly relates, in a preferred embodiment, to characterization of polymer samples in a comprehensive, directly-coupled, multi-dimensional high-performance liquid chromatography systems including a first HPLC dimension adapted for determining composition (e.g., adapted for reverse phase chromatography, adsorption chromatography, and the like such as mobile phase gradient-elution chromatography) and a second HPLC dimension adapted for determining molecular weight or size (e.g., adapted for size exclusion chromatography such as gel permeation chromatography).
Multi-dimensional high-performance liquid chromatography systems are known in the art. See e.g., Murphy et al., Effect of Sampling Rate on Resolution in Comprehensive Two-Dimensional Liquid Chromatography, Anal. Chem. 70, 1585-1594 (1998); Murphy et al., One-and Two-Dimensional Chromatographic Analysis of Alcohol Ethoxylates, Anal. Chem. 70, 4353-4360 (1998); Kilz et al., Two Dimensional Chromatography for the Deformulation of Complex Copolymers, Chapter 17, pp. 223-241 of the text entitled xe2x80x9cChromatographic Characterization of Polymers, Hyphenated and Multidimensional Techniquesxe2x80x9d, edited by Provder et al. (American Chemical Society, Advances in Chemistry Series 247, 1995); Opiteck et al., Two-Dimensional SEC/RPLC Coupled to Mass Spectrometry for the Analysis of Peptides, Anal. Chem. 69, 2283-2291 (1997); and Trathnigg et al., Two-Dimensional Liquid Chromatography of Functional Polyethers, Chapter 13, pp. 190-199 of the text entitled xe2x80x9cChromatography of Polymers, Hyphenated and Multidimensional Techniquesxe2x80x9d, edited by Provder et al. (American Chemical Society, Symposium Series 731, 1999), each of which is hereby incorporated by reference for all purposes.
Although the methods and systems disclosed to date in the art have proven to be useful for characterizing biological and non-biological polymer samples, they generally suffer from inefficiencies with respect to overall sample throughput, and/or with respect to complicated control and/or operation schemes and systems.
Accordingly, there remains a need in the art for improved methods and systems for effecting multi-dimensional liquid chromatography for characterization of polymer samples.
It is therefore an object of the present invention to provide methods and apparatus that allow for more efficient, and relatively less complicated approaches than the prior art for characterizing polymer samples, and especially for fingerprinting polymer samples such as non-biological copolymer samples.
Briefly, therefore, the present invention is directed, generally, to methods for characterizing a polymer sample in a multi-dimensional liquid chromatography system. In preferred embodiments, the invention is directed to methods for characterizing a library of polymer samples in a multi-dimensional liquid chromatography system. The multi-dimensional liquid chromatography system comprises at least a first dimension and a second dimension, and in some embodiments, can include a third dimension, a fourth dimension and/or additional dimensions. Preferably, each of the first dimension and the second dimension is a high-performance liquid chromatography (HPLC) subsystem. The multi-dimension liquid chromatography system is preferably a comprehensive multi-dimension liquid chromatography system wherein at least a portion of each of the sample components separated in the first dimension are further separated into subcomponents in the second dimension. Further, the first dimension and second dimension of the multi-dimensional liquid chromatography system are preferably directly-coupled, wherein the components separated in the first dimension are sampled in near real time (e.g., in-line) as they elute off of the first-dimension chromatography column(s) for injection into the second dimensionxe2x80x94for example, through a second-dimension in-line multi-port injection valve.
The method generally comprises, for characterization of a single polymer sample, injecting the polymer sample into a first-dimension high-performance liquid chromatography subsystem, separating the polymer sample into two or more components in the first-dimension liquid chromatography subsystem, optionally detecting a property of the first-dimension separated components in the first-dimension eluent (e.g., using a flow-through detector), sampling at least a portion of each of the first-dimension separated components for directly-coupled injection into a second dimension, injecting each of the sampled portions into a second-dimension high-performance liquid chromatography subsystem, separating at least one of, and preferably each of the sampled portions of the first-dimension separated components into two or more subcomponents in the second-dimension liquid chromatography subsystem, and detecting a property of the second-dimension separated subcomponents in the second-dimension eluent (e.g., using a flow-through detector).
More specifically, for characterizing a single polymer sample, the polymer sample is injected (e.g., using a multi-port injection valve as a first-dimension injector) into a first-dimension mobile phase of a first HPLC dimension of the multi-dimensional liquid chromatography system. At least one sample component of the injected polymer sample is chromatographically separated from other sample components thereof in a first-dimension liquid chromatography column (e.g., in selectable fluid communication with the first-dimension injector), such that a first-dimension mobile phase eluent from the first-dimension column comprises two or more first-dimension separated sample components. Optionally, a property of the first-dimension separated components in the first-dimension mobile phase effluent can be detected using a flow-through detector (e.g. mass detector, universal concentration detector, light-scattering detector, etc.). Then, at least a portion of each of the first-dimension separated sample components from the first-dimension mobile phase eluent are sampled for directly-coupled injection into a second HPLC dimension of the multi-dimensional chromatography system (e.g., using sample loops associated with a multi-port injection valve). The sampled portions of each of the first-dimension separated sample components are then injected directly into a second-dimension mobile phase of the second HPLC dimension of the multi-dimensional liquid chromatography system (e.g., using a multi-port injection valve as a second-dimension injector). At least one subcomponent of the injected sample portions is chromatographically separated from other subcomponents thereof in a second-dimension liquid chromatography column (e.g., in selectable fluid communication with the second-dimension injector), such that a second-dimension mobile phase eluent from the second-dimension column comprises two or more second-dimension separated subcomponents for one or more, and in some cases, for each of the sampled portions of each of the first-dimension separated sample components. A property of the second-dimension separated subcomponents are detected in the second-dimension mobile phase effluent using a flow-through detector.
For characterization of a library of polymer samples comprising four or more polymer samples, the aforementioned steps, as generally or specifically characterized, of injecting into the first dimension, separating into components in the first dimension, optionally detecting separated components in the first-dimension eluent, injecting into the second dimension, separating into subcomponents in the second dimension and detecting separated subcomponents in the second-dimension eluent are repeated for each of the polymer samples of the library.
In preferred embodiments, the method is further characterized according to one or more of the following characterizing embodiments, considered independently or in combination in any of the various possible permutations.
In a first characterizing embodiment, at least a portion of each of the first-dimension separated sample components are sampled by repetitively sampling discrete volumes of the first-dimension mobile phase eluent at regularly recurring time intervals. That is, the sampling for the second dimension is effected at regular, recurring intervals of time without regard to whether or not a first-dimension separated component of the sample is present and actually sampled. Advantageously, such an approach is relatively less complicated than other schemes for second-dimension sampling, is robust, and has universal applicability across a wide range of polymers. Moreover, by controlling the separation rates of both the first and second dimensions (e.g., with the overall separation rate being characterized, for example, as the injection rate into the first dimension), together with controlling the second-dimension sampling frequency and sample size, high-resolution multi-dimensional characterization can be effected.
In a second characterizing embodiment, the second-dimension of the multi-dimensional liquid chromatography system is a parallel-column high-performance liquid chromatography subsystem, with serially-selected or parallel detection. More specifically, the second dimension of the multi-dimensional liquid chromatography system comprises two or more parallel second-dimension liquid chromatography columns, and a second-dimension mobile phase is continuously supplied in parallel through the two or more second-dimension liquid chromatography columns. The sampled portions of the first-dimension separated sample components are serially and distributively injected into the second-dimension mobile phases of the two or more second-dimension liquid chromatography columns, respectively. At least one subcomponent of the injected sample portions is then chromatographically separated from other subcomponents thereof substantially simultaneously (i.e., slightly offset temporally) in the respective second-dimension liquid chromatography columns. Advantageously, such an approach provides for substantially improved overall sample throughput, since the multiple second-dimension samples can be substantially simultaneously evaluated, with a relatively uncomplicated mechanical system comprising a single common injector. Moreover, effecting the chromatographic separation step of the second dimension in parallel (i.e., substantially simultaneous separation using two or more second-dimension columns) can advantageously provide a significant improvement of the second dimension resolution by allowing for relatively prolonged second dimension separation times for each of the sampled portions of the first-dimension eluent (as compared to a strictly serial second-dimension chromatographic separation and analysis), while keeping the overall number of second dimension separations the same as can be effected in the serial second-dimension separation. Generally, the operational conditions of the first and second dimensions can be selected to achieve an appropriate balance between the overall sample throughput (in the first and/or second dimension) and the desired resolution.
A third characterizing embodiment is directed to a method for characterizing a library of polymer samples. In this embodiment, a library of polymer samples are provided for characterization in the multi-dimensional liquid chromatography system, with the library comprising four or more different polymer samples for analysis. The multi-dimensional liquid chromatography system comprises a first dimension and a second dimension, with one of the first or second dimensions being adapted for size exclusion chromatography. In a particularly preferred embodiment, the second dimension HPLC subsystem is adapted for size-exclusion chromatography (SEC) such as gel permeation chromatography (GPC). More specifically, in this third characterizing embodiment, at least a portion of each of the first-dimension separated sample components are sampled by sampling at least ten discrete volumes of the first-dimension mobile phase eluent. The steps of injecting a polymer sample into the first-dimension mobile-phase, chromatographically separating the injected polymer in the first dimension, optionally detecting a property of the first-dimension separated components, sampling the first-dimension mobile phase eluent for injection into the second-dimension, injecting into the second dimension, separating in the second dimension, and detecting a property of the second-dimension separated subcomponents are repeated for each of the four or more polymer samples of the library, with the four or more polymer samples of the library being successively injected into the first-dimension mobile phase of the first dimension at intervals of not more than about 30 minutes per sample. In preferred approaches for this embodiment, the injection-to-injection interval is preferably not more than about 15 minutes, and more preferably not more than about 10 minutes.
The present invention is directed as well, to an apparatus for effecting the above-identified methods. That is, the invention is directed as well to multi-dimensional liquid chromatography systems comprising a-first dimension high-performance liquid chromatography subsystem and a second dimension high-performance liquid chromatography subsystem. In general, the first dimension HPLC subsystem comprises a first-dimension mobile phase source in fluid communication with a first-dimension liquid chromatography column, a first-dimension pump in fluid communication with the first dimension mobile phase source and with the first-dimension column for continuously supplying a first-dimensional mobile phase through the first dimension column, an injection valve in selectable fluid communication with the first-dimension mobile phase for serially injecting polymer samples into the first-dimension mobile phase, and optionally, a first-dimension flow-through detector in fluid communication with the first-dimension mobile phase eluent for detecting a property of the first-dimension separated sample component. The second dimension HPLC subsystem comprises a second-dimension mobile phase source in fluid communication with a second-dimension liquid chromatography column, a second-dimension pump in fluid communication with the second dimension mobile phase source and with the second-dimension column for continuously supplying a second-dimensional mobile phase through the second dimension column, a second-dimension injector in selectable fluid communication with the first-dimension mobile phase eluent and in selectable fluid communication with the second-dimension mobile phase for serially sampling at least a portion of the first-dimension separated components from the first-dimension mobile phase eluent and for injecting the sampled portion into the second-dimension mobile phase, and a second-dimension flow-through detector in fluid communication with the second-dimension mobile phase eluent for detecting a property of the second-dimension separated subcomponents.
In preferred embodiments, the multi-dimensional liquid chromatography systems are further characterized according to one or more of the following characterizing embodiments, considered independently or in combination in any of the various possible permutations.
In one characterizing embodiment, the multi-dimensional liquid chromatography system is further characterized as comprising a controller for the second-dimension injector, the controller being adapted for sampling discrete volumes of the first-dimension mobile phase eluent at regularly recurring time intervals, and for injecting the sampled volumes into the second-dimension mobile phase.
In another characterizing embodiment, the multi-dimensional liquid chromatography system is further characterized as having a first-dimension HPLC subsystem comprising a single mobile phase analysis channel, and a second-dimension HPLC subsystem comprising at least two analysis channels in parallel. More specifically, the second-dimension HPLC subsystem comprises at least two second-dimension liquid chromatography columns, and is adapted to continuously supply the second-dimension mobile phase in parallel through the two or more second-dimension liquid chromatography columns (e.g., from the second-dimension mobile phase source). In preferred aspects of this characterizing embodiment, the second-dimension mobile-phase is supplied to each of the second-dimension columns through one or more flow restrictors.
In yet a further characterizing embodiment, the multi-dimensional liquid chromatography system is further characterized as comprising a control system adapted for serially injecting successive polymer samples into the first dimension mobile phase of the system at intervals of not more than about 30 minutes for sample, and adapted for sampling at least ten discrete volumes of the first-dimensional mobile phase eluent, and injecting the at least ten sampled volumes directly into the second dimension mobile phase.
In particularly preferred embodiments, including both method embodiments and apparatus embodiments, the following features can be applied generally with respect to any of the aforementioned embodiments, alone or in combination in the various permutations. Generally, the polymer samples being characterized can be non-biological polymers (e.g., non-biological copolymers) or biological polymers (e.g., proteins, DNA), and in many applications, are preferably non-biological polymers. Generally, the first dimension HPLC subsystem can be adapted for chromatographic approaches effective for distinguishing between chemical composition and/or structural variations of polymer sample components (e.g., repeat units types, ratios of copolymer repeat units, functional groups, branching, etc.). Exemplary preferred first-dimension HPLC subsystems include reverse phase chromatography subsystems, mobile-phase compositional gradient elution chromatography subsystems, or mobile-phase temperature gradient elution chromatography subsystems. Mobile-phase elution gradients of the first dimension preferably comprise a substantially universal co-solvent system, such as a water-tetrahydrofuran-hexane system. Generally, the second dimension HPLC subsystem is preferably adapted for size-exclusion chromatography (SEC) such as gel permeation chromatography (GPC). Additionally, the flow-through detector of the second dimension HPLC subsystem is generally preferably a universal concentration detector or mass detector, such as an evaporative light-scattering detector (ELSD). Further, generally, the first and second dimension liquid chromatography subsystems can be combined with further dimensions, such as third, forth or higher dimensions, and such further dimensions can be liquid-chromatography subsystems, gas-chromatography subsystems, electrophoretic subsystems, electrochromatographic subsystems, field-flow fractionation subsystems, flow-injection analysis subsystems, or other types of polymer characterization systems, such as mass spectrometry.
The methods and apparatus of the invention are particularly useful for characterizing individual polymer samples, or libraries of polymer samples, such as non-biological polymer samples, and especially for characterizing combinatorial libraries of polymers (e.g. synthesized using parallel polymerization approaches). The methods and apparatus of the invention can be advantageously applied for polymer fingerprintingxe2x80x94determining both compositional/structural characteristics as well as molecular size/molecular weight characteristics. The methods and apparatus of the invention can also be used for effective scale up of a polymerization synthesis process, to ensure that the fingerprint of the polymer made by large-scale synthesis process is substantially the same as the polymer made by the smaller scale synthesis process.
The methods and apparatus of the invention can be applied using convention, macro-scale liquid chromatography systems, or alternatively, can be applied in a micro-scale or nano-scale format, such as in microfluidic devices such as lab-on-a-chip liquid microfluidic chromatography devices.
Other features, objects and advantages of the present invention will be in part apparent to those skilled in art and in part pointed out hereinafter. All references cited in the instant specification are incorporated by reference for all purposes. Moreover, as the patent and non-patent literature relating to the subject matter disclosed and/or claimed herein is substantial, many relevant references are available to a skilled artisan that will provide further instruction with respect to such subject matter.